Portal de Periódicos da CAPES

Expression of the Integrin-Linked Kinase (ILK) in Mouse Skin

1998; Elsevier BV; Volume: 153; Issue: 2 Linguagem: Inglês

10.1016/s0002-9440(10)65580-0

1525-2191

Wen Xie, Fugang Li, Jeffrey E. Kudlow, Chuanyue Wu,

Wound Healing and Treatments

Integrin-linked kinase (ILK) is a newly identified serine/threonine protein kinase implicated in integrin signaling. To investigate the functions of ILK in vivo, we have analyzed the expression and regulation of ILK in the skin, in which proper control of cell-extracellular matrix interactions and cell proliferation is essential for its normal development and homeostasis. We report here that ILK is abundantly expressed throughout the extracellular matrix-rich dermis. ILK mRNA was also detected in the hair follicles and the basal cells of the interfollicular epidermis. However, ILK expression is lost in the suprabasal layers of keratinocytes that are undergoing terminal differentiation. PINCH, an ILK-binding protein, exhibited a similar expression pattern in the skin. Recent studies have indicated that erbB-2, a member of the epidermal growth factor receptor family, plays a pivotal role in epidermal growth, differentiation, and hair follicle morphogenesis. Using a transgenic mouse system in which an activated erbB-2 is overexpressed in the epidermis, we show that ILK expression is regulated by erbB-2. The in vivo expression and regulation patterns of ILK, together with its biochemical activities, suggest an important role of ILK in coordinating the integrin signaling pathways and the growth factor signaling pathways in the development of the skin and the pathogenesis of skin diseases. Integrin-linked kinase (ILK) is a newly identified serine/threonine protein kinase implicated in integrin signaling. To investigate the functions of ILK in vivo, we have analyzed the expression and regulation of ILK in the skin, in which proper control of cell-extracellular matrix interactions and cell proliferation is essential for its normal development and homeostasis. We report here that ILK is abundantly expressed throughout the extracellular matrix-rich dermis. ILK mRNA was also detected in the hair follicles and the basal cells of the interfollicular epidermis. However, ILK expression is lost in the suprabasal layers of keratinocytes that are undergoing terminal differentiation. PINCH, an ILK-binding protein, exhibited a similar expression pattern in the skin. Recent studies have indicated that erbB-2, a member of the epidermal growth factor receptor family, plays a pivotal role in epidermal growth, differentiation, and hair follicle morphogenesis. Using a transgenic mouse system in which an activated erbB-2 is overexpressed in the epidermis, we show that ILK expression is regulated by erbB-2. The in vivo expression and regulation patterns of ILK, together with its biochemical activities, suggest an important role of ILK in coordinating the integrin signaling pathways and the growth factor signaling pathways in the development of the skin and the pathogenesis of skin diseases. Integrin-linked kinase (ILK) is a newly identified serine/threonine protein kinase that has been implicated in cellular control of cell-extracellular matrix interactions and cell proliferation.1Hannigan GE Leung-Hagesteijn C Fitz-Gibbon L Coppolino MG Radeva G Filmus J Bell JC Dedhar S Regulation of cell adhesion and anchorage-dependent growth by a new β1-integrin-linked protein kinase.Nature. 1996; 379: 91-96Crossref PubMed Scopus (955) Google Scholar, 2Radeva G Petrocelli T Behrend E Leunghagesteijn C Filmus J Slingerland J Dedhar S Overexpression of the integrin-linked kinase promotes anchorage-independent cell cycle progression.J Biol Chem. 1997; 272: 13937-13944Crossref PubMed Scopus (218) Google Scholar, 3Wu C Keightley SY Leung-Hagesteijn C Radeva G Coppolino M Goicoechea S McDonald JA Dedhar S Integrin-linked protein kinase (ILK) regulates fibronectin matrix assembly, E-cadherin expression, and tumorigenicity.J Biol Chem. 1998; 273: 528-536Crossref PubMed Scopus (252) Google Scholar ILK regulates integrin-mediated cell adhesion, E-cadherin expression, and extracellular fibronectin matrix assembly.1Hannigan GE Leung-Hagesteijn C Fitz-Gibbon L Coppolino MG Radeva G Filmus J Bell JC Dedhar S Regulation of cell adhesion and anchorage-dependent growth by a new β1-integrin-linked protein kinase.Nature. 1996; 379: 91-96Crossref PubMed Scopus (955) Google Scholar, 3Wu C Keightley SY Leung-Hagesteijn C Radeva G Coppolino M Goicoechea S McDonald JA Dedhar S Integrin-linked protein kinase (ILK) regulates fibronectin matrix assembly, E-cadherin expression, and tumorigenicity.J Biol Chem. 1998; 273: 528-536Crossref PubMed Scopus (252) Google Scholar Moreover, overexpression of ILK in rat epithelial cells induces anchorage-independent cell growth in culture1Hannigan GE Leung-Hagesteijn C Fitz-Gibbon L Coppolino MG Radeva G Filmus J Bell JC Dedhar S Regulation of cell adhesion and anchorage-dependent growth by a new β1-integrin-linked protein kinase.Nature. 1996; 379: 91-96Crossref PubMed Scopus (955) Google Scholar and tumor formation in vivo.3Wu C Keightley SY Leung-Hagesteijn C Radeva G Coppolino M Goicoechea S McDonald JA Dedhar S Integrin-linked protein kinase (ILK) regulates fibronectin matrix assembly, E-cadherin expression, and tumorigenicity.J Biol Chem. 1998; 273: 528-536Crossref PubMed Scopus (252) Google Scholar Biochemical and cell biological analyses have shown that ILK is intimately involved in the cell adhesion-dependent cell cycle progression by regulating the level and/or activity of several key components of cell cycle machinery, including cyclin A, cyclin D1, and cyclin-dependent kinases.2Radeva G Petrocelli T Behrend E Leunghagesteijn C Filmus J Slingerland J Dedhar S Overexpression of the integrin-linked kinase promotes anchorage-independent cell cycle progression.J Biol Chem. 1997; 272: 13937-13944Crossref PubMed Scopus (218) Google Scholar In addition to binding to integrins, we recently have found that ILK interacts with PINCH (Y Tu, F Li, S Goicoechea, C Wu, manuscript submitted for publication), an intracellular adaptor protein comprising primarily five LIM domains.4Rearden A A new Lim protein containing an autoepitope homologous to "senescent cell antigen.".Biochem Biophys Res Commun. 1994; 201: 1124-1131Crossref PubMed Scopus (74) Google Scholar The ILK gene has been mapped to the human chromosome 11p15.5 to p15.4 region5Hannigan GE Bayani J Weksberg R Beatty B Pandita A Dedhar S Squire J Mapping of the gene encoding the integrin-linked kinase, ILK, to human chromosome 11p15.5–p15.4.Genomics. 1997; 42: 177-179Crossref PubMed Scopus (40) Google Scholar and a conserved region in mouse chromosome 7,6Li F Liu J Mayne R Wu C Identification and characterization of a mouse protein kinase that is highly homologous to human integrin-linked kinase.Biochim Biophys Acta. 1997; 1358: 215-220Crossref PubMed Scopus (25) Google Scholar which includes a syntenic group of genes encoding Harvey-ras, insulin-like growth factor II, and target of antiproliferative antibody 1. It is particularly interesting to note that several breakpoints associated with the Beckwith-Wiedemann syndrome, a genetic disorder with overgrowth and predisposition to Wilms' tumor, are located in the same area within the human chromosome 11p15.5 region.7Shows TB Alders M Bennett S Burbee D Cartwright P Chandrasekharappa S Cooper P Courseaux A Davis C Devignes M-D Deville P Elliott R Evans G Fantes J Garner H Gaudray P Gerhard DS Gessler M Higgins M Hummerich H James M Lagercrantz J Litt M Little P Mannens M Munroe D Nowak N O'Brien S Parker N Perlin M Reid L Richard C Sawicki M Swallow D Thakker R van Heyningen V van Schothorst E Vorechovsky I Wadelius C Weber B Zabel B Report of the Fifth International Workshop on Human Chromosome 11 Mapping (1996).Cytogenet Cell Genet. 1996; 74: 1-56Crossref PubMed Scopus (57) Google Scholar Although the biochemical, cell biological, and genetic evidence has implicated important roles of ILK in the pathogenesis of tumor and other hyperproliferative diseases, the in vivo expression and regulation of ILK were previously not known. Previous studies have shown that members of the epidermal growth factor receptor family play critical roles in cell differentiation and proliferation in the skin.8Miettinen PJ Berger JE Meneses J Phung Y Pedersen RA Werb Z Derynck R Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor.Nature. 1995; 376: 337-341Crossref PubMed Scopus (862) Google Scholar, 9Sibilia M Wagner EF Strain-dependent epithelial defects in mice lacking the EGF receptor [published erratum appears in Science 1995, 269: 909].Science. 1995; 269: 234-238Crossref PubMed Scopus (849) Google Scholar, 10Threadgill DW Dlugosz AA Hansen LA Tennenbaum T Lichti U Yee D LaMantia C Mourton T Herrup K Harris RC Barnard JA Yuspa SH Coffey RJ Magnuson T Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype.Science. 1995; 269: 230-234Crossref PubMed Scopus (1252) Google Scholar, 11Murillas R Larcher F Conti CJ Santos M Ullrich A Jorcano JL Expression of a dominant negative mutant of epidermal growth factor receptor in the epidermis of transgenic mice elicits striking alterations in hair follicle development and skin structure.EMBO J. 1995; 14: 5216-5223Crossref PubMed Scopus (240) Google Scholar, 12Hansen LA Alexander N Hogan ME Sundberg JP Dlugosz A Threadgill DW Magnuson T Yuspa SH Genetically null mice reveal a central role for epidermal growth factor receptor in the differentiation of the hair follicle and normal hair development.Am J Pathol. 1997; 150: 1959-1975PubMed Google Scholar, 13Xie W Chow LT Wu X Chin E Paterson AJ Kudlow JE Targeted expression of activated erbB-2 to the epidermis of transgenic mice elicits striking developmental abnormalities in the epidermis and hair follicles.Cell Growth Differ. 1998; 9: 313-325PubMed Google Scholar For example, overexpression of a constitutively active form of erbB-2, a member of the epidermal growth factor receptor family,14Dougall WC Qian X Peterson NC Miller MJ Samanta A Greene MI The neu-oncogene: signal transduction pathways, transformation mechanisms and evolving therapies.Oncogene. 1994; 9: 2109-2123PubMed Google Scholar, 15Carraway KLR Cantley LC A neu acquaintance for erbB3 and erbB4: a role for receptor heterodimerization in growth signaling.Cell. 1994; 78: 5-8Abstract Full Text PDF PubMed Scopus (582) Google Scholar in the hair follicles and the basal cells of the epidermis resulted in profound epidermal, dermal, and hair follicle abnormalities, including epidermal hyperplasia, preneoplasia, papilloma, hyperkeratosis, dyskeratosis, and dermal hyperplasia.13Xie W Chow LT Wu X Chin E Paterson AJ Kudlow JE Targeted expression of activated erbB-2 to the epidermis of transgenic mice elicits striking developmental abnormalities in the epidermis and hair follicles.Cell Growth Differ. 1998; 9: 313-325PubMed Google Scholar The majority of the hair follicles were replaced by bizarre hyperproliferative intradermal squamous invaginations, whereas the rest of the follicles exhibited severe hyperplasia and disorganization.13Xie W Chow LT Wu X Chin E Paterson AJ Kudlow JE Targeted expression of activated erbB-2 to the epidermis of transgenic mice elicits striking developmental abnormalities in the epidermis and hair follicles.Cell Growth Differ. 1998; 9: 313-325PubMed Google Scholar It becomes clear now that the growth factor signaling pathways coordinate with the integrin signaling pathways in control of cell proliferation and differentiation.16Brooks PC Klemke RL Schon S Lewis JM Schwartz MA Cheresh DA Insulin-like growth factor receptor cooperates with integrin αvβ5 to promote tumor cell dissemination in vivo.J Clin Invest. 1997; 99: 1390-1398Crossref PubMed Scopus (154) Google Scholar, 17Miyamoto S Teramoto H Gutkind JS Yamada KM Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: roles of integrin aggregation and occupancy of receptors.J Cell Biol. 1996; 135: 1633-1642Crossref PubMed Scopus (673) Google Scholar, 18Fujii K Ligand activation of overexpressed epidermal growth factor receptor results in loss of epithelial phenotype and impaired RGD-sensitive integrin function in HSC-1 cells.J Invest Dermatol. 1996; 107: 195-202Crossref PubMed Scopus (8) Google Scholar, 19Plopper GE McNamee HP Dike LE Bojanowski K Ingber DE Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex.Mol Biol Cell. 1995; 6: 1349-1365Crossref PubMed Scopus (467) Google Scholar To investigate the functions of ILKin vivo, we have analyzed the expression of ILK in normal mouse skins and those of the erbB-2-transgenic mice. We report here that ILK is expressed by the dermal fibroblasts, the outer root sheath cells of the hair follicles, and the basal cells of the epidermis in normal mouse skins. The ILK expression is lost in the differentiating and postmitotic suprabasal keratinocytes. Strikingly, overexpression of the activated erbB-2 dramatically and specifically increased ILK expression along the basal layers of the hyperplastic epidermis, the squamous invaginations, and the outer root sheath-equivalent compartment of the hyperplastic follicles. These results provide important in vivo evidence suggesting a role of ILK in regulating cell proliferation and differentiation in the skin. Normal mice (B6 × SJL) were obtained from the animal facility of the University of Alabama at Birmingham. The K14-erbB-2 transgenic mice were generated by injection of one-cell B6 × SJL mouse zygotes with a K14-erbB-2 transgene in which the cDNA encoding an activated form of rat c-neu/erbB-2 (with Val664 to Glu664 mutation)20Bargmann CI Weinberg RA Oncogenic activation of the neu-encoded receptor protein by point mutation and deletion.EMBO J. 1988; 7: 2043-2052Crossref PubMed Scopus (270) Google Scholar, 21Bargmann CI Hung MC Weinberg RA Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185.Cell. 1986; 45: 649-657Abstract Full Text PDF PubMed Scopus (806) Google Scholar was placed downstream of the 2.3-kb promoter for the human K14 gene.13Xie W Chow LT Wu X Chin E Paterson AJ Kudlow JE Targeted expression of activated erbB-2 to the epidermis of transgenic mice elicits striking developmental abnormalities in the epidermis and hair follicles.Cell Growth Differ. 1998; 9: 313-325PubMed Google Scholar All mice were handled in the University of Alabama at Birmingham animal facility in accordance with the institutional animal care policies. Frozen sections (10 μm) of the back skins from normal neonatal (1-day-old) B6 × SJL mice and the K14-erbB-2 transgenic mice were subjected to in situ hybridization using35S-labeled anti-sense riboprobes that correspond to the full-length mouse ILK cDNA (1.4 kb),6Li F Liu J Mayne R Wu C Identification and characterization of a mouse protein kinase that is highly homologous to human integrin-linked kinase.Biochim Biophys Acta. 1997; 1358: 215-220Crossref PubMed Scopus (25) Google Scholar human PINCH cDNA (0.9 kb),4Rearden A A new Lim protein containing an autoepitope homologous to "senescent cell antigen.".Biochem Biophys Res Commun. 1994; 201: 1124-1131Crossref PubMed Scopus (74) Google Scholar and the C-terminal fragment of the rat erbB-2 cDNA (2.7-kb NdeI-SalI fragment),13Xie W Chow LT Wu X Chin E Paterson AJ Kudlow JE Targeted expression of activated erbB-2 to the epidermis of transgenic mice elicits striking developmental abnormalities in the epidermis and hair follicles.Cell Growth Differ. 1998; 9: 313-325PubMed Google Scholar respectively. High-specific activity riboprobes were synthesized in 10-μl reactions containing 100 μCi [35S]UTP and 100 μCi [35S]CTP (Amersham, Arlington Heights, IL), 10 mmol/L NaCl, 6 mmol/L MgCl2, 40 mmol/L Tris (ph 7.5), 2 mmol/L spermidine, 10 mmol/L dithiothreitol, 500 μmol/L each of unlabeled ATP and GTP, 25 μmol/L each of unlabeled UTP and CTP, 0.5–1 μg linearized template, 15 U of the appropriate polymerase, and 15 U RNase inhibitor (RNasin, Promega, Madison, WI). The reaction mixtures were incubated at 42°C for 60 minutes. Labeled cRNA probes were purified with Bio-Spin 6 columns (Bio-Rad, Richmond, CA). The skin sections were hybridized with the probes at 55°C temperature for 15 to 18 hours.13Xie W Chow LT Wu X Chin E Paterson AJ Kudlow JE Targeted expression of activated erbB-2 to the epidermis of transgenic mice elicits striking developmental abnormalities in the epidermis and hair follicles.Cell Growth Differ. 1998; 9: 313-325PubMed Google Scholar After hybridization, emulsion dip (NTB2 nuclear emulsion, Kodak, Rochester, NY), exposure, and developing, the sections were counterstained with hematoxylin and eosin and subjected to microscopic examination under bright-field and dark-field illumination. In control experiments, serial sections of the back skins from both the normal and K14-erbB-2 transgenic mice were hybridized with 35S-labeled cRNA probes for hexaminidase A and O-linked N-acetyl glucosamine transferase, respectively. The results showed that hexaminidase A and O-linked N-acetyl glucosamine transferase were expressed by both epidermal and dermal cells. Moreover, in contrast to ILK expression, which was markedly increased in the K14-erbB-2 transgenic mouse skins (see Results), neither the expression of hexaminidase A nor that of O-linked N-acetyl glucosamine transferase was altered by the overexpression of the activated erbB-2. Paraffin sections of the erbB-2-transgenic and control mouse back skins were stained with a mouse monoclonal anti-PCNA antibody (1:100 dilution, BioGenex, San Ramon, CA). After washing, the bound anti-PCNA antibody was detected with a biotinylated goat anti-mouse IgG antibody (Vector Laboratories, Burlingame, CA). The immune complexes were visualized with a Vectastain Elite ABC Kit (Vector Laboratories) using 3,3′-diaminobenzidine tetrahydrochloride as chromogen. The sections were slightly counterstained with Gill's hematoxylin (Vector Laboratories). We analyzed the expression of ILK in mouse skins by in situ hybridization. The skin sections from the back of a 1-day-old normal B6 × SJL mouse were hybridized with a35S-labeled antisense cRNA probe of ILK. Abundant ILK mRNA was detected in the outer root sheath cells of the hair follicles and in the dermal fibroblasts (Figure 1, A(bright field) and B (dark field)). Additionally, ILK mRNA was present, although at a relatively lower level, in the basal cells of the interfollicular epidermis. In contrast, no appreciable ILK expression was detected in the spinous and granular layers or in the stratum corneum. We next determined the cellular expression of the ILK-binding protein PINCH in the skin. The skin sections of the neonatal mouse were hybridized with a 35S-labeled antisense cRNA probe of PINCH. The results showed that PINCH mRNA was primarily expressed by the dermal and the outer root sheath cells (Figure 1, C (bright field) and D (dark field)). No PINCH expression was detected in the suprabasal keratinocytes of the epidermis. Thus, the expression pattern of PINCH mRNA resembles that of ILK, consistent with a role of PINCH in ILK function in vivo. To determine whether ILK expression in the skin is altered under pathological conditions, we analyzed the ILK expression in the skins of the K14-erbB-2 transgenic mice. In normal mouse skins, erbB2 is expressed predominantly in the outer root sheath of the hair follicles.13Xie W Chow LT Wu X Chin E Paterson AJ Kudlow JE Targeted expression of activated erbB-2 to the epidermis of transgenic mice elicits striking developmental abnormalities in the epidermis and hair follicles.Cell Growth Differ. 1998; 9: 313-325PubMed Google Scholar, 22Kokai Y Cohen JA Drebin JA Greene MI Stage- and tissue-specific expression of the neu oncogene in rat development.Proc Natl Acad Sci USA. 1987; 84: 8498-8501Crossref PubMed Scopus (137) Google Scholar, 23Quirke P Pickles A Tuzi NL Mohamdee O Gullick WJ Pattern of expression of c-erbB-2 oncoprotein in human fetuses.Br J Cancer. 1989; 60: 64-69Crossref PubMed Scopus (57) Google Scholar, 24Maguire Jr, HC Jaworsky C Cohen JA Hellman M Weiner DB Greene MI Distribution of neu (c-erbB-2) protein in human skin.J Invest Dermatol. 1989; 92: 786-790Abstract Full Text PDF PubMed Google Scholar In addition, low levels of erbB-2 transcripts were also present throughout the interfollicular epidermis with relatively higher expression in the basal cells.13Xie W Chow LT Wu X Chin E Paterson AJ Kudlow JE Targeted expression of activated erbB-2 to the epidermis of transgenic mice elicits striking developmental abnormalities in the epidermis and hair follicles.Cell Growth Differ. 1998; 9: 313-325PubMed Google Scholar, 22Kokai Y Cohen JA Drebin JA Greene MI Stage- and tissue-specific expression of the neu oncogene in rat development.Proc Natl Acad Sci USA. 1987; 84: 8498-8501Crossref PubMed Scopus (137) Google Scholar, 23Quirke P Pickles A Tuzi NL Mohamdee O Gullick WJ Pattern of expression of c-erbB-2 oncoprotein in human fetuses.Br J Cancer. 1989; 60: 64-69Crossref PubMed Scopus (57) Google Scholar, 24Maguire Jr, HC Jaworsky C Cohen JA Hellman M Weiner DB Greene MI Distribution of neu (c-erbB-2) protein in human skin.J Invest Dermatol. 1989; 92: 786-790Abstract Full Text PDF PubMed Google Scholar The K14 promoter faithfully targets the expression of the transgene (activated erbB-2) to the basal cells and the outer root sheath of the hair follicles, sites to which the endogenous erbB-2 expression has been localized.13Xie W Chow LT Wu X Chin E Paterson AJ Kudlow JE Targeted expression of activated erbB-2 to the epidermis of transgenic mice elicits striking developmental abnormalities in the epidermis and hair follicles.Cell Growth Differ. 1998; 9: 313-325PubMed Google Scholar The K14-erbB-2 transgenic mice exhibited severe epidermal hyperplasia, hyperplastic hair follicle, and numerous hyperproliferative intradermal squamous invaginations that may arise from abnormally developed hair follicles.13Xie W Chow LT Wu X Chin E Paterson AJ Kudlow JE Targeted expression of activated erbB-2 to the epidermis of transgenic mice elicits striking developmental abnormalities in the epidermis and hair follicles.Cell Growth Differ. 1998; 9: 313-325PubMed Google Scholar The back skin sections of neonatal K14-erbB-2 transgenic mice were hybridized with a 35S-labeled ILK cRNA probe (Figure 2, A (bright field) B (dark field)) or a 35S-labeled erbB-2 cRNA probe (Figure 2, C(bright field) and D (dark field)) as a control. Abundant erbB-2 mRNA was detected along the basal layers of the hyperplastic epidermis and squamous invaginations of the transgenic skins (Figure 2, C and D), consistent with the basal and the outer root sheath specificity of the K14 promoter.25Vassar R Fuchs E Transgenic mice provide new insights into the role of TGF-α during epidermal development and differentiation.Genes Dev. 1991; 5: 714-727Crossref PubMed Scopus (367) Google Scholar Strikingly, ILK expression was significantly and specifically up-regulated in the erbB-2-overexpressing basal-most several layers of the hyperplastic squamous invaginations (Figure 2, A and B), whereas the ILK expression in dermal fibroblasts remained unchanged. Therefore, the expression of ILK along the basal layers is distinctively higher than the dermal fibroblasts, which markedly differs from the skin of the normal mouse, in which ILK is equally expressed in the hair follicles and dermal fibroblasts (Figure 1, A and B). Staining of the transgenic and control mouse skin sections with an anti-PCNA26Bravo R Frank R Blundell PA Macdonald-Bravo H Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta.Nature. 1987; 326: 515-517Crossref PubMed Scopus (1636) Google Scholar antibody, a marker of proliferating cells,27Hall PA Levison DA Woods AL Yu CC Kellock DB Watkins JA Barnes DM Gillett CE Camplejohn R Dover R Waseem NH Lane DP Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms.J Pathol. 1990; 162 (see comments): 285-294Crossref PubMed Scopus (1395) Google Scholar showed that the cells in which ILK expression was up-regulated by erbB-2 overexpression were highly proliferative (Figure 2F). Additionally, we have observed that the expression of PINCH was also increased, although to a much lesser extent than that of the ILK expression, in the areas where the activated erbB-2 was overexpressed (eg, the outer root sheath of the hair follicles; data not shown). The results obtained from this study reveal several important features of ILK expression in the skin. First, ILK expression in normal mouse epidermis is confined to the outer root sheath of hair follicles and the basal keratinocytes of the interfollicular epidermis. The keratinocytes in the suprabasal layers, which are undergoing terminal differentiation and are largely postmitotic, are devoid of ILK. The highly restricted expression pattern of ILK in the epidermis resembles that of the β1 integrins, to which ILK binds.1Hannigan GE Leung-Hagesteijn C Fitz-Gibbon L Coppolino MG Radeva G Filmus J Bell JC Dedhar S Regulation of cell adhesion and anchorage-dependent growth by a new β1-integrin-linked protein kinase.Nature. 1996; 379: 91-96Crossref PubMed Scopus (955) Google Scholar The β1 integrins normally are also confined to the proliferative basal keratinocytes.28Watt FM Jones PH Expression and function of the keratinocyte integrins.Development. 1993; suppl: 185-192Google Scholar Watt and Jones have shown that the proliferative potential of the keratinocytes is correlated to the level and the activation state of the cell-surface β1 integrins,28Watt FM Jones PH Expression and function of the keratinocyte integrins.Development. 1993; suppl: 185-192Google Scholar and the stem cells can be effectively isolated based on their strong adhesion to fibronectin, type IV collagen, or other extracellular matrix proteins.29Jones PH Watt FM Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression.Cell. 1993; 73: 713-724Abstract Full Text PDF PubMed Scopus (986) Google Scholar Moreover, forced expression of functional β1 integrins (eg, α5β1 or α2β1) in the suprabasal epidermal layers of transgenic mice resulted in epidermal hyperproliferation, perturbed keratinocyte differentiation, and other features reminiscent of psoriasis.30Carroll JM Romero MR Watt FM Suprabasal integrin expression in the epidermis of transgenic mice results in developmental defects and a phenotype resembling psoriasis.Cell. 1995; 83: 957-968Abstract Full Text PDF PubMed Scopus (284) Google Scholar The similarity between the expression patterns of ILK and the β1 integrins, together with the previous observations that ILK is capable of binding and phosphorylating the β1 integrins1Hannigan GE Leung-Hagesteijn C Fitz-Gibbon L Coppolino MG Radeva G Filmus J Bell JC Dedhar S Regulation of cell adhesion and anchorage-dependent growth by a new β1-integrin-linked protein kinase.Nature. 1996; 379: 91-96Crossref PubMed Scopus (955) Google Scholar and promoting integrin-mediated fibronectin matrix assembly,3Wu C Keightley SY Leung-Hagesteijn C Radeva G Coppolino M Goicoechea S McDonald JA Dedhar S Integrin-linked protein kinase (ILK) regulates fibronectin matrix assembly, E-cadherin expression, and tumorigenicity.J Biol Chem. 1998; 273: 528-536Crossref PubMed Scopus (252) Google Scholar suggest that ILK likely works in concert with the β1 integrins in regulation of keratinocyte proliferation and differentiation. The second important observation of this study is that ILK expression is regulated by erbB-2, an oncogenic protein, in vivo. ErbB-2 is normally expressed in basal cells of the epidermis and the outer root sheath of the hair follicles.22Kokai Y Cohen JA Drebin JA Greene MI Stage- and tissue-specific expression of the neu oncogene in rat development.Proc Natl Acad Sci USA. 1987; 84: 8498-8501Crossref PubMed Scopus (137) Google Scholar, 23Quirke P Pickles A Tuzi NL Mohamdee O Gullick WJ Pattern of expression of c-erbB-2 oncoprotein in human fetuses.Br J Cancer. 1989; 60: 64-69Crossref PubMed Scopus (57) Google Scholar, 24Maguire Jr, HC Jaworsky C Cohen JA Hellman M Weiner DB Greene MI Distribution of neu (c-erbB-2) protein in human skin.J Invest Dermatol. 1989; 92: 786-790Abstract Full Text PDF PubMed Google Scholar, 31Press MF Cordon-Cardo C Slamon DJ Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissues.Oncogene. 1990; 5: 953-962PubMed Google Scholar Overexpressing the constitutively active erbB-2 under the control of human keratin K14 promoter, which directed its expression to cells in which erbB-2 is normally expressed, induced extensive and striking hyperplastic skin phenotypes13Xie W Chow LT Wu X Chin E Paterson AJ Kudlow JE Targeted expression of activated erbB-2 to the epidermis of transgenic mice elicits striking developmental abnormalities in the epidermis and hair follicles.Cell Growth Differ. 1998; 9: 313-325PubMed Google Scholar that share many features that, although more severe, are similar to those observed in the K14-TGFα transgenic mice.25Vassar R Fuchs E Transgenic mice provide new insights into the role of TGF-α during epidermal development and differentiation.Genes Dev. 1991; 5: 714-727Crossref PubMed Scopus (367) Google Scholar All but one of the K14-erbB-2 transgenic mice died before or shortly after birth, probably as a consequence of defects in the skin and esophagus.13Xie W Chow LT Wu X Chin E Paterson AJ Kudlow JE Targeted expression of activated erbB-2 to the epidermis of transgenic mice elicits striking developmental abnormalities in the epidermis and hair follicles.Cell Growth Differ. 1998; 9: 313-325PubMed Google Scholar It has become increasingly clear that the integrin signaling pathways coordinate with the growth factor pathways in regulation of cell proliferation. In fact, the hair follicle and eyelid defects induced by overexpression of the β1 integrins are extremely similar to those induced by mutations in transforming growth factor α32Mann GB Fowler KJ Gabriel A Nice EC Williams RL Dunn AR Mice with a null mutation of the TGF α gene have abnormal skin architecture, wavy hair, and curly whiskers and often develop corneal inflammation.Cell. 1993; 73: 249-261Abstract Full Text PDF PubMed Scopus (520) Google Scholar, 33Luetteke NC Qiu TH Peiffer RL Oliver P Smithies O Lee DC TGF α deficiency results in hair follicle and eye abnormalities in targeted and waved-1 mice.Cell. 1993; 73: 263-278Abstract Full Text PDF PubMed Scopus (574) Google Scholar or epidermal growth factor receptor.34Luetteke NC Phillips HK Qiu TH Copeland NG Earp HS Jenkins NA Lee DC The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase.Genes Dev. 1994; 8: 399-413Crossref PubMed Scopus (387) Google Scholar Because ILK is involved in integrin signaling and can activate several key components of cell cycle machinery,2Radeva G Petrocelli T Behrend E Leunghagesteijn C Filmus J Slingerland J Dedhar S Overexpression of the integrin-linked kinase promotes anchorage-independent cell cycle progression.J Biol Chem. 1997; 272: 13937-13944Crossref PubMed Scopus (218) Google Scholar the increase of ILK expression induced by overexpression of the activated erbB-2 in the epidermis likely contributes to the extensive and striking hyperplastic skin abnormalities, including epidermal hyperplasia, preneoplasia, papilloma, hyperkeratosis, dyskeratosis, and dermal hyperplasia, observed in the erbB-2 transgenic mice.13Xie W Chow LT Wu X Chin E Paterson AJ Kudlow JE Targeted expression of activated erbB-2 to the epidermis of transgenic mice elicits striking developmental abnormalities in the epidermis and hair follicles.Cell Growth Differ. 1998; 9: 313-325PubMed Google Scholar It will be important to determine in future studies whether overexpression of ILK in the epidermis can induce a phenotype similar to that of the erbB-2 transgenic mice. Abundant ILK expression in normal mouse skin was detected throughout the dermis, into which extensive extracellular matrices were deposited. Previous studies have shown that integrins not only receive signals from the extracellular matrix but also actively participate in the formation of the extracellular matrix.35Wu C Keivens VM TE OT McDonald JA Ginsberg MH Integrin activation and cytoskeletal interaction are essential for the assembly of a fibronectin matrix.Cell. 1995; 83: 715-724Abstract Full Text PDF PubMed Scopus (294) Google Scholar, 36Wu C Roles of integrins in fibronectin matrix assembly.Histol Histopathol. 1997; 12: 233-240PubMed Google Scholar In a recent study, we found that overexpression of ILK in cultured intestinal epithelial cells, which normally assemble littler fibronectin matrix, dramatically stimulated the deposition of fibronectin into the matrix.3Wu C Keightley SY Leung-Hagesteijn C Radeva G Coppolino M Goicoechea S McDonald JA Dedhar S Integrin-linked protein kinase (ILK) regulates fibronectin matrix assembly, E-cadherin expression, and tumorigenicity.J Biol Chem. 1998; 273: 528-536Crossref PubMed Scopus (252) Google Scholar Thus, loss of ILK expression in the suprabasal layers of the epidermis is likely functionally important for keratinocyte terminal differentiation, as the terminal differentiation of keratinocytes is inhibited in the presence of fibronectin.37Adams JC Watt FM Fibronectin inhibits the terminal differentiation of human keratinocytes.Nature. 1989; 340: 307-309Crossref PubMed Scopus (315) Google Scholar On the other hand, the high expression level of ILK in the matrix-rich dermis may reflect, among other things, a positive regulatory role of ILK in promoting extracellular matrix deposition in vivo. In summary, our results provide in vivo evidence supporting a role of ILK in regulation of cell proliferation and cell-matrix interactions in the skin. Future studies should include analyses of ILK expression and regulation in human hyperproliferative and fibrotic skin diseases.